A standardized method for plasma extracellular vesicle isolation and size distribution analysis

The following protocol describes our workflow for isolation and quantification of plasma extracellular vesicles (EVs). It requires limited sample volume so that the scientific value of specimens is maximized. These steps include isolation of vesicles by automated size exclusion chromatography and quantification by tunable resistive pulse sensing. This workflow optimizes reproducibility by minimizing variations in processing, handling, and storage of EVs. EVs have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. This standardized protocol is scalable and ensures efficient recovery of physiologically intact EVs that may be used in a variety of downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely. Absolute quantification corrects for variations in EV number and size, offering a novel method of standardization in downstream applications.

This protocol details the measurement of EVs from plasma samples. All human subjects research should be reviewed and approved by the site-specific Institutional Review Board (IRB) prior to proceeding. This protocol uses the IZON qNano Gold TRPS instrument. If other TRPS measurement systems are used (e.g. IZON Exoid), this workflow will likely need to be adjusted slightly to incorporate alternate equipment.
Additional Notes: Additional Notes: 1. 2x PBS is preferred for measuring exosomes (30-150 nm) because it decreases the background noise. 2. Strategies for nanopore opening: These troubleshooting strategies can be used in any combination to facilitate the stabilization or opening of a nanopore. These methods can also be used to help unclog a nanopore when a run is paused.

Tapping
Tapping → With the VPM nozzle in place, use your index finger to firmly tap the shielding cap 3-5 times. This can disrupt bubbles and assist with pore opening.

Clicking
Clicking → Remove pressure from system and open pressure valve. Remove VPM nozzle. Raise the shielding and set it on the nanopore such that the posts of the shielding cap are resting on the arms of the nanopore. Apply downward pressure and begin to rotate until the shielding cap abruptly "clicks" downward into place.
Pressure application device (PAD) use Pressure application device (PAD) use → Remove pressure from system and open pressure valve. Remove VPM nozzle and the shielding cap. Center PAD over upper fluid cell and give 10 plunges. Repeat as necessary to unclog nanopore or reduce RMS noise. Nanopore cleaning Nanopore cleaning → Remove pressure from system and open pressure valve. Remove VPM nozzle. Remove shielding cap and upper fluid cell. Clean upper fluid cell with filtered water and dry completely. Reduce stretch on nanopore to 42 mm and remove from posts. Rinse each side of nanopore with 500 μL 70% ethanol followed by 500 μL filtered water. Carefully dry each side of nanopore with edge of Kimwipe. Dry lower fluid cell with Kimwipe. Replace nanopore on the posts and rotate knob clockwise to achieve desired stretch. Pipet 75 μL 2x ME carefully into the lower fluid cell, avoiding the introduction of bubbles. Replace upper fluid cell and shielding cap. Carefully pipette 35 μL 2x ME into upper fluid cell and click "Turn On". Monitor current and RMS noise, proceed with other troubleshooting strategies as necessary.
Adjusting pore stretch Adjusting pore stretch → once a pore has been used, it is possible that it will need to be stretched >47 mm. Test nanopore stability at increased stretch values (maximum recommended stretch is 48 mm). Appropriate personal protective equipment (PPE) should be worn (nitrile gloves, safety goggles, and lab coat). Store all organic solvents in a flammable storage cabinet in accordance with institution policy. Utilize biohazard waste containers for sample waste.
Ensure IZON software has been installed on computer that will be used to run qNano instrument.
Reagents should be room temperature when used on the qNano. Allow adequate acclimation to ambient temperature prior to stretching the nanopore or beginning the experiment. Thaw sample(s) at Room temperature Room temperature and centrifuge 10000 x g 10000 x g for 00:10:00 00:10:00 .
We recommend preparing and measuring two samples simultaneously given our experience with sample run time and nanopore stability.
We utilized a 10 cm 10 cm tissue culture dish to keep nanopore off benchtop and to collect cleaning fluids.

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Rinse nanopore with 500 µL 500 µL filtered water to remove ethanol. Pat dry with kimwipe, careful not to apply pressure to the central nanopore.

11
Rinse bottom electrode with 75 µL 75 µL filtered water and dry completely with kimwipe.
Proceed immediately to next step. 13.1 Measure distance between three-prong posts opposite one another.
13.2 Once the initial stretch is confirmed and recorded, the software will track additional adjustments.
13.3 Begin recording the duration of time that the nanopore has been stretched.
Nanopores typically begin to destabilize or overstretch when used >6-7 hours.
14 To wet the nanopore, slowly pipet 75 µL 75 µL 2x ME into the lower fluid cell being careful not to introduce bubbles.
The lower fluid cell is the space between the bottom electrode and the bottom surface of the nanopore. Bubbles will contribute to significant background noise during measurement.

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Clean the upper fluid cell with 500 µL 500 µL filtered water and dry completely by tapping firmly on a kimwipe or drying directly with a kimwipe.

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Fit the upper fluid cell on the qNano above the nanopore and add 35 µL 35 µL 2x ME into the central well of the upper fluid cell, being careful to avoid introducing bubbles. opening" for strategies to promote pore opening and stabilization.
It is unlikely that the pore will open completely without tapping/clicking. An expected stable baseline current at 0.10 V is 10-38 nA.
20 Close the pressure valve and increase the pressure with the pressure bar to 8 mbar-10 mbar.
21 Slowly increase the voltage in a step-wise manner until a current of 110-135 nA is reached.
We recommend targeting 120 nA for a working current. The RMS noise should be <10. If it is above 10, refer to "Strategies for pore opening". 23 Clean upper fluid cell by tapping firmly on kimwipe and/or drying with kimwipe directly.
24 Gently use the edge of a kimwipe to dry the top of the nanopore.
25 Gently use the edge of a kimwipe to dry the top of the nanopore. 27 Replace VPM nozzle, close pressure valve, and set pressure bar to 3 mbar ('Working Pressure 1').
The actual pressure reading in the software will be slightly different from the pressure set with the pressure bar. It is recommended to start at lower pressures for smaller pores, such as the NP100.
28 Observe particle size and adjust pore stretch and voltage accordingly.
If current is higher/lower than 120 nA, adjust voltage such that the current is 116-124 nA. Particle rate should increase with increased pressure. If this does not occur, the nanopore is not appropriately stabilized or the sample preparation is inadequate. The user may try higher pressures, differently diluted sample, or nanopore cleaning procedures.

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Record 2-3 technical replicates per sample and average replicates post-hoc for more accurate concentration estimates.
We found that this was not typically necessary as the recordings were highly similar, however it may be helpful when troubleshooting samples. Sample changeover procedures do not need to followed when recording technical replicates.
Nanopore and qNano Cleaning Nanopore and qNano Cleaning 51 Click "Turn off" to discontinue current across the nanopore.
52 Remove pressure by pulling out pressure bar completely followed by opening the pressure valve.

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Remove the nanopore and rinse the nanopore from both sides with 500 µL 500 µL 70% ethanol followed by 500 µL 500 µL deionized water. Gently dry with clean with kimwipe and secure Nanopore in respective pouch and record date and usage hours on pouch.
Cleaning the pore as described is only necessary if the nanopore will be used again. If the nanopore is at the maximum stretch time, it can be discarded in biohazard waste.